Cell culture and mixing vessel

ABSTRACT

A mixing vessel ( 10 ) for containing a liquid, comprises a chamber having a lower chamber portion and an upper chamber portion wider than the lower portion, gas inlet means ( 14 ) for supplying gas to the lower portion and means for redirecting rising gas ( 24 ), such that, in use, rising gas in the form of bubbles, initially rises substantially vertically and is redirected in a substantially horizontal direction by the means for redirecting rising gas.

This application is a continuation of U.S. Application Ser. No.11/639,413, filed Dec. 15, 2006and claims priority benefit of GB0525579.9, filed Dec. 16, 2005 and GB 0617490.8, filed Sep. 5, 2006

TECHNICAL FIELD

The invention relates to a mixing vessel suitable for carrying out acell culture, particularly an aerobic cell culture.

BACKGROUND AND PRIOR ART

Many academic and industrial processes utilise cell culture to generatecells or in the production of biomaterials or compounds of interest.

In other processes, such as ultra filtration or the mixing of biologicalsolutions or the dissolution of solids into aqueous solutions, it isnecessary to provide fast, effective and yet gentle mixing of the liquidmedium.

In cell culture, an aliquot of cells is placed in a vessel of somedescription, provided with the nutrients required for the growth of thecells, and is either supplied with oxygen or grown anaerobically in theabsence of oxygen. After a period of time to allow production and growthof new cells, the cultured cells are typically removed from a vessel andharvested or separated from the medium.

In processes upstream and/or downstream from the culture process, it isoften important to mix cells, media or other materials, to prevent themfrom settling in a storage tank or from forming precipitates. In theseinstances, it is essential to keep the medium contained within thevessel from settling and so it is advantageous for the medium in thevessel to be kept in constant motion.

Since the first use of aerobic cell culture systems, there have been aseries of physical constraints that workers in the field have attemptedto address. One of the primary constraints is how to achieve goodaeration of the culture without incurring (excessive) damage to thecells being grown and at the same time to achieve a homogenoussuspension, or mixing, of cells and to avoid the formation of unstirredareas or “dead spots”.

One arrangement widely used in cell culture employs sparging air in thebottom of a vessel so as to cause recirculation of the culture medium.This further improves aeration without the need for physical agitationand has the added benefit of mixing the cell culture without the needfor expensive stirring, rolling or rocking devices. The type of culturesystem that uses an internal air-powered aeration system is oftendescribed as a pressure-cycle fermenter or airlift fermenter.

In a pressure-cycle fermenter, the aerated stream of gas and solutionthat results from sparging the vessel at its base, has a lower densitythan that of the undisturbed solution and therefore rises. This streamis often referred to as the “riser”. The solution that is drawn in totake its place forms a stream, often referred to the “downcomer”.Circulation within the cell culture can therefore be achieved simply byaerating the culture as described in GB2002417A, U.S. Pat. No. 5,660,977and GB 1383432A.

However this method often suffers from having regions of ‘dead zones’ inwhich the culture media is not effectively mixed. To overcome thisproblem, many inventions have sought to separate the riser and thedowncomer through the use of a physical barrier. GB2002417A, U.S. Pat.No. 5,660,977 and GB1383432A describe a variety of configurations inwhich this is achieved. Generally, the riser is directed to the surfaceof the culture solution through a tube that is arranged vertically andruns from the bottom, up to just below the surface of the culturemedium. The riser moves upwards, and is replaced by the downcomer whichcirculates the solution. In other arrangements, the downcomer can be anexternal tube.

Other arrangements. such as that disclosed in U.S. Pat. No. 4,649,117,do not require the use of a separation device to divide the riser andthe downcomer. In this arrangement, the upper surface of the culturemedium is considerably wider than at the base, which contains thesparger. While this will generate the required riser and downcomer, itcan be observed in such systems that there are often areas of theculture that are mixed less well than others to give “dead spots” or“flat spots”. To compensate for this, the operator will often increasethe aeration rate to achieve better mixing. This is a disadvantagebecause increased sparging will also give rise to the requirement formore gas, which increases process cost, and greater foaming. Foamingmust be controlled, as for certain mammalian cell culture systems, thereis a direct correlation between increased sparge rate and increased cellmortality. This trend can be entirely eliminated by controlling foamingthrough the addition of antifoaming agents. One skilled in the art willrecognize that a better solution to this problem is to keep foaming to aminimum so as to reduce the cost of the antifoam addition or thepossible interference of the antifoam reagents in downstream processes.

JP61202680A teaches that cells attached to a carrier (generally a beadwith an activated surface to which cells adhere) can be kept insuspension in a chamber that is separated from the media feed and mixingapparatus by a porous plate that allows the passage of media and gas butdoes not allow the carriers, with their adherent cells, from passingthrough the porous separator plate. This is an advantage because thebeads are kept separate from the stirring means, which tend to fragmentthe beads and therefore loose their ability to bind cells when damaged.However, the porous plate cannot separate cells that are grown in freesuspension, which would therefore pass through the porous plate.Therefore JP61202680A is limited in the type of cells which can besuccessfully grown, and prevent it from being applied in the growth ofcells such as insect cells, yeast and bacteria, all of which are grownin suspension and not on surfaces.

The costs of assembling and maintaining such fermentation and cellculture systems in a production environment has always been high andsteps have therefore been taken to develop disposable systems made fromlow-cost plastics. EP 0 343 885 discloses a system that utilises thepressure-cycle principle in a plastics bag, in which the riser anddowncomer are separated by an intervening plastics sheet, to promote thepressure-cycle effect. The plastics vessel is sparged using a tubeinserted from the top of the plastics vessel and the air is dispersedinto small air bubbles through the use of a metallic frit assembly.While the use of a separating sheet may in theory provide bettercirculation due to the pressure-cycle process, overall, it reduces theamount of mixing that takes place and inevitably causes “dead zones” asmentioned above. A very similar arrangement of a reusable plastics bagwith a downward-pointing sparging pipe was described in US 2002/0110915A1 in which no separator was employed.

Many of the commercially available cell culture systems have relied onthe use of some form of impeller, under which the aerating gas isdelivered into the cell culture. The speed at which the impeller isrotated, its shape and surface area all have the potential to improvethe rate of oxygen transfer. A good summary of the art of impellerdesign can be found in U.S. Pat. No. 6,250,797. One of the majordisadvantages of agitation with impeller blades of any shape is thatthey cause shear stress and often lead to excessive foaming of the cellculture. Considerable heat can also be generated which must be removed,further increasing costs for production. These issues are to be avoidedwith non-bacterial cells as this results in lower biomass production andpoor cell viability.

Other methods of improving oxygen transfer rates without inducing shearstress as a result of deploying impellers have been developed. Forexample, flexible plastics bags provide a number of opportunities foralternative means of gentle agitation. Bags can be manipulated throughthe use of external pressure applied mechanically (U.S. Pat. No.3,819,158), by moving liquid from one bag chamber to another (EP 972 506A), from rocking the bag back-and-forth (U.S. Pat. No. 6,190,913) or bymoving one portion of the bag using mechanical means with pneumaticcollars or cuffs (US 2005/0063250 A). In these cases, it is still oftennecessary to enrich the airflow into the air space above the culture inorder to achieve sufficient rates of oxygen transfer and such systemsare only used to grow cells with a low requirement for oxygen.

These issues are not confined to cell culture, but also apply in thepreparation of solutions and solids that are used in bio production ofall types, or in the isolation of the bio product from the cell culturemedium. The use of a disposable modules in such operations significantlyreduces the cost of cleaning and revalidation of the plant's componentscompared with modules made from glass and stainless steel. However, theprovision of effective mixing has remained a major challenge for many ofthe reasons already discussed. Agitation must be achieved either byrecalculating a liquid at great velocity, which itself causes problems,or through the inclusion of a disposable propeller driven directlythrough a sealed bearing system (which must be disposable) or throughthe use of a magnetically coupled and disposable propeller. Bothgenerate heat, excessive shear forces and are expensive.

SUMMARY OF INVENTION

The invention provides a mixing vessel for containing a liquid,comprising a chamber having a lower chamber portion and an upper chamberportion wider than the lower portion, gas inlet means for supplying gasto the tower portion and redirecting means for redirecting rising gas,such that in use, rising gas in the form of bubbles, initially risessubstantially vertically in liquid and is redirected in a substantiallyhorizontal direction by the redirecting means for redirecting risinggas.

Gas introduced into the lower chamber portion rises through the liquid.As the chamber is wider in the upper chamber portion than in the lowerchamber portion, introduced gas, preferably sterile gas, rises onlythrough a partial volume of the liquid. This gassified partial volumehas a reduced density in relation to the liquid which is not subject tothe rising gas. This difference in density can cause liquid in the upperchamber portion to circulate within the upper chamber portion as aresult of the introduced gas. Thus circulation pathways which existcompletely within the upper chamber portion can be established.

The chamber has a redirecting means for redirecting rising gas so thatthe rising gas in the form of bubbles is deflected by the redirectingmeans, and is redirected in a substantially horizontal direction. Thismovement significantly enhances the circulation within the upper chamberas well as gently disturbing the surface of the liquid, which improvesgas transport between the headspace and the liquid surface, whencompared with vessels that do not possess this feature.

Conveniently, the redirecting means comprises an abutment inclined withrespect to the direction of the rising gas.

The shape of the vessel therefore gives both good aeration of the liquidand mixing of the liquid without the need for mechanical agitation andprovides a high surface area of liquid in contact with the headspaceabove the liquid for further gaseous exchange.

In another aspect, the invention provides a mixing vessel for containinga liquid. comprising a chamber having a lower chamber portion and anupper chamber portion wider than the lower portion, gas inlet means forsupplying gas to the lower portion and redirecting means for redirectingrising gas comprising an abutment inclined with respect to the directionof rising gas, such that, in use, rising gas in the form of bubbles,initially rises substantially vertically in liquid and is redirected ina substantially horizontal direction by the abutment.

In another aspect, the invention provides a cell culture vesselcomprising a chamber having a lower chamber portion and an upper chamberportion wider than the lower portion and gas inlet means for supplyinggas to the lower portion, such that, in use, liquid in the upper chamberportion circulates within the upper chamber portion as a result of theintroduced gas.

As well as liquid circulation within the upper chamber portion, someliquid preferably also travels downwardly from the upper chamber portioninto the lower chamber portion in a downcomer for subsequent upwardsflow under the action of rising gas from the inlet means.

Geometry of Vessel

The chamber typically comprises a back wall, at least a part of which isvertical or substantially vertical (e.g. ±10°), and a front wall. Thehorizontal distance between the front wall and back wall is referred toas the width of the chamber, which varies with height to constitute thewider upper chamber portion and the narrower lower chamber portion. Theback and front walls are typically linked by two side walls which areusually vertical or substantially vertical.

Where the vessel comprises an abutment, it is conveniently inclinedrelative to the vertical or substantially vertical part of the backwall. In a preferred embodiment, the abutment is a non-vertical part ofthe back wall. Alternatively the abutment may be a separate componentfixed with respect to the vessel.

The gas inlet means preferably extends across substantially the wholelength of the back wall and is such that the gas rises close to the backwall. The circulation within the upper chamber portion then typicallytakes place away from the back wall in a widened region and has atendency to establish a tubular circulation pattern about a horizontalaxis parallel to the back wall.

Thus, in view of the geometry of the vessel, excellent mixing propertiesare obtained without the need for physical barriers between risingregions of liquid and descending regions of liquid. Thus, preferably anysuch physical barriers are not present.

The volume of the chamber which is filled with liquid in use is referredto herein as the working volume. The space above the liquid in thechamber constitutes a headspace.

The fill level of the vessel must be selected so that the means forredirecting rising gas, e.g. an abutment, can operate effectively.

Preferably at least part of the back wall is planar for reasons ofsimplicity of construction. A planar wall has the additional advantagethat it can be readily placed in contact with or close to a temperatureregulation plate.

The widening of the vessel with respect to height preferably occursasymmetrically. In other words it is preferable that the lower chamberportion is not centrally located below the upper chamber portion. Thisfurther differentiates the current invention from U.S. Pat. No.4,649,117and others, in which the vessel is essentially symmetrical andusually cylindrical.

Preferably the lower chamber portion is tapered, becoming wider withincreasing height, typically having a front wall inclined with respectto the back wall. The angle of taper is conveniently in the range of 5°to 40°, preferably 10° to 30°, e.g. about 20°. This tapered arrangementincreases the amount of circulating liquid in the downcomer re-enteringthe gassified partial volume. Preferably the lower chamber portion is sotapered that it is not undesirably wide at the base thereof. Ideally thelower chamber portion has a width such that the gas inlet means can fitsnugly at the base of the vessel. This helps to prevent any dead zonesfrom forming near the base.

Preferably at least a lower part of the upper chamber portion is alsotapered, preferably at a more gradual incline than the lower chamberportion, so as to give sufficient width to the upper chamber. Thisarrangement may be achieved by appropriately varying the angle ofinclination of the front wall. The angle of taper is conveniently in therange of from 25° to 80°, preferably 40° to 70°, e.g. 60°. An upper partof the upper chamber is desirably more steeply tapered than the lowerpart, with the front wall possibly being vertical or even turning backon itself. This arrangement assists with prevention of dead zones andfurther improves circulation. In other words, the angle of incline ofthe front wall, has an initial value and with increasing height isfollowed by a reduction in the incline of at least 0°, preferably atleast 20°, which in turn is followed by an increase in the incline of atleast 0°, preferably at least 20°. The reduction and increase may begradual or sudden.

The ratio of the height of the working volume of the chamber to themaximum width of the working volume of the chamber (the aspect ratio)should be appropriately selected to give both good circulation andaeration. A tall chamber has the advantage of having a high hydrostaticpressure at the base as well as a long rise height to the surface forthe introduced gas to travel along. Both of these improve the rate oftransport of gas into the liquid. An effective chamber should also havesufficient width in the upper chamber portion to provide enough volumefor bulk circulation in the upper chamber portion. As a firstapproximation, the aspect ratio should be no less than 0.5. An aspectratio of greater than 1.0 is preferred.

If the surface area of liquid in the vessel is A, the rise height of thegas H and the volume of liquid is V, then G=(H×A) I V is preferablygreater than 1, preferably greater than 1.2, more preferably greaterthan 1.5. A typical maximum value of G is 4, although it is preferablyless than 3. Ideally G has a value of about 2.

The vessel may be made out of any suitable material, such as glass,metallic materials or polymeric materials, typically being made ofglass, steel or plastics materials.

When made out of flexible material, the inner bag may be held on orwithin a suitable rigid support, or be supported by anchoring the bag tothe vessel by a suitable support, although usually, the inner bag isheld in position when enclosed by the vessel through a combination ofgas pressure inside the bag and the addition of the aqueous solutioninto the bag.

One skilled in the art will appreciate that the principle of theinvention can be applied to vessels with, or without an inner liner bag.

One of the key advantages of the vessel's geometry is that it can be inthe form of a disposable bag, typically being supported by a framefitting the geometry of the vessel. The bag's contents are typicallyisolated from the environment through the use of suitable filters andmay contain an integrated sparging tube that is arranged from left toright along its base. The bag effectively acts as a “liner” beingintended for single use and made with plastics materials well suited forthis purpose. Thereby, the bag can be replaced, without the supportingframe ever coming into contact with the solutions that the bag contains.In this instance the supporting frame imposes a physical constraint onthe flexible bag such that the bag adopts the geometry of the vessel.Advantageously, the bag is slightly oversized so that it pushes into anycorners when inflated. The bag is desirably provided in a sterile,pre-validated condition and sealed in packaging. Advantageously, the bagmay be made from one or multiple layers of plastics materials which arepreferably co-extruded during manufacture and preferably under cleanroomconditions. In this case, preferably at least one of the layers exhibitsa high resistance to gas diffusion e.g. nylon or EVA. Another one ofthese layers may be made from polyethylene which exhibits highresistance to aqueous solution. Preferably any plastics materials usedare free from materials derived from animal sources. In a preferredembodiment co-extruded layers are used with the polyethylene layer on aninner face and the other layer(s) on an outer face.

The vessel is typically sealed at the top to prevent ingress of anythingwhich may interfere with a cell culture process or the materials beingmixed. The vessel is optionally fitted with a filter, which preventsingress of external gas and particulates while allowing gas from theairspace to be vented. The gas outlet assembly optionally comprises apressure regulator, set such that pressure in the headspace may becontrolled. Alternatively the headspace may comprise a pressure sensor,the reading of which may be fed to a computer which can be programmed tocontrol the air inlet rate to maintain a fixed headspace pressure.

The vessel may also have one or more additional inlet and outlet ports,arranged at any convenient point around the vessel, e.g. in theheadspace or just above the gas inlet means. Any additional inlet andoutlet ports may be used for introduction of liquid nutrients or foradditional gases or for removing samples. Adding nutrients just abovethe gas inlet means has the advantage that they will be readily mixedwith the bulk of the liquid by the rising gas stream.

Gas Inlet Means

The gas inlet means introduces gas in the form of bubbles which riseupwards through the liquid culture medium in use of the vessel, or theinner bag if this option is adopted. The gas inlet means preferably hasa plurality of exit holes which generate a plurality of gas bubbles. Theexit holes may be constituted by pores of a porous material such asceramic material, silicon, porous polymer etc., or by apertures in asolid material such as metal, plastics etc. The size of the exit holesin the gas inlet means conveniently range from 0.1 microns to 1 mm,preferably from 1 to 100 microns. The size of bubbles generated rangesfrom 0.1 to 2 mm. In a preferred embodiment

The gas inlet means delivers gas to the lower chamber portion at anyconvenient location. Preferably gas is introduced as close to the baseof the vessel as possible, although from experimental observations, itsprecise location does not appear to reduce the efficiency of mixing aslong as it is located within the lower quarter of the vessel's height.

It has been found that a gas inlet means which introduces gas over anelongate region is particularly effective at mixing and gaseousexchange. Such an arrangement gives a rising wall of bubbles. This maybe achieved by a single elongate gas inlet means with exit holes alongits length. Alternatively a sequence of separate inlet devices could beused to the same effect. Because of the tendency of rising bubbles tospread out, an elongate region of rising bubbles could even be achievedwhen the separate inlet devices are relatively widely spaced. Indeed,experimental observations indicate that effective mixing occurs when thesparging tube occupies no more than 15% of the vessel's width. Separatedinlet devices may share a single gas supply if arranged in series, orcan each have their own gas supply if arranged in parallel.

The gas inlet means conveniently comprises one or more tubes with gaspermeable walls. The shortest distance between tube and the back walland the shortest distance between the tube and the front wall arepreferably both less than 3 times the diameter of the tube. Preferablyboth distances are no greater than the diameter of the tube.

In a preferred embodiment, the gas inlet means is elongate and made ofmedical grade polyethylene with a pore size of 20 to 40 microns.

The vessel may be accompanied by a separate controller device, which maybe capable of monitoring and controlling various operating parameterssuch as flow rate, oxygen concentration, headspace pressure etc.

The invention also provides mixing apparatus, particularly cell cultureapparatus, including a vessel in accordance with the invention. Theapparatus suitably includes appropriate control means, e.g. in the formof a separate controller device.

Use of the Vessel

It has been found to be advantageous to operate a cell culture in thevessel of the present invention with an elevated headspace pressure.This can be achieved by having a means for providing a controllablepressure in gas above the liquid, e.g. by suitable selection of apressure relief valve and inlet gas flow rate. An elevated pressure hasbeen found to improve the rate of transport of gas into solution. Anygas in the headspace will transfer into the liquid across the topsurface of the liquid more rapidly at higher pressure. Additionally therising gas will also experience the elevated pressure, giving furtherimprovement in gas transport. It is believed that an elevated pressurealso helps to prevent or reduce undesirable foam generation at theliquid surface, which is sometimes encountered in cell culturefermentation. A headspace pressure of from 0.01 to 10 psi gauge,preferably from 0.1 to 2 psi gauge conveniently gives the aboveadvantages.

In another aspect, the invention provides a method of performing a cellculture fermentation in a vessel having an enclosed headspace, themethod comprising operating at a headspace pressure of from 0.01 to 10psi gauge, preferably from 0.1 to 2 psi gauge.

Typically the gas to be fed through the gas inlet means contains oxygen.Conveniently compressed air can be fed into the vessel through the gasinlet means, however some cell types may have a greater demand foroxygen than can be delivered with air alone, and so an oxygen-enrichedgas may be utilised. The inlet gas is typically sterile.

The inlet gas is normally fed at a continuous steady rate, however forcertain applications it may be advantageous to pulse the gas throughdiscontinuously. The rate of pulsing may be selected so as to giveoptimal mixing. Computer control may be used to regulate intermittentgas flow.

If the vessel has additional gas inlet ports, then a gas of similar ordifferent composition to the gas introduced through the gas inlet meansmay be used. For example an oxygen/carbon dioxide mixture can be passedthrough the inlet means in the lower chamber portion and oxygen could beintroduced directly into the headspace. This combination would bebeneficial for situations where depiction of carbon dioxide is to beavoided whilst maintaining an oxygen rich head space above the liquid soas to give effective oxygenation. A person skilled in the art willappreciate that many other combinations are possible.

The same means for introducing gases for aeration of cell culturesystems and the efficient mixing that this generates can also be used tomix other aqueous and non-aqueous solutions, fluids, liquids and solidsand suspensions. The ability to gently mix a vessel in this way is aconsiderable advantage over stirred or rocked systems as it can bereadily scaled-up to nearly any working volume, as one skilled in theart will appreciate.

A means for regulating the temperature of the liquid in the vessel ispreferably provided. Typically the temperature will be required to bemaintained constant within the range of from 5 to 40° C., preferablyfrom 16 to 37° C. for cell culture. Temperature regulation can beimplemented by a variety of techniques as will be known to a personskilled in the art of cell culture fermentation. Conveniently,temperature regulation can be achieved by use of a temperatureregulation plate in contact with or close to the back wall of thevessel, as mentioned above.

Although the vessel of the present invention has good mixing propertiesand does not require mechanical agitation, mechanical agitation mayadditionally be employed if desired or required. This could for exampletake the form of part of a flexible vessel wall being intermittentlydeformed, e.g. depressed It is conceivable that a separate impellermeans may be present inside the vessel, although this may introduceunhelpful shear forces and is ideally avoided. External manipulation ofan inner liner bag may be useful in mixing suspensions of particularlyviscous materials.

The geometry of the present invention gives effective mixing andaeration over a wide range of sizes and can, for example, be used with aliquid culture volume of from 1 to 10,000 liters. The vessel may bemaintained through manual intervention or under computer control.Suitable sensor means may be provided to monitor appropriate parametersto provide information for control purposes. The invention lends itselfparticularly well to the use of non-invasive probes with whichmeasurements are recorded by following a fluorescence-based reporter,attached to the inner surface of the vessel such as that taught in U.S.Pat. Nos. 5,037,615 and 5,152,287.

The invention will be further described, by way of illustration, in theaccompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic side sectional view of a preferred embodiment ofvessel according to the invention;

FIG. 2 is a schematic rear view of the vessel shown in FIG. 1;

FIG. 3 is a simplified schematic perspective view of the vessel of FIGS.1 and 2;

FIG. 4 is a schematic perspective view illustrating another embodimentof a vessel according to the invention;

FIG. 5 is another schematic perspective view illustrating a furtherembodiment of a vessel according to the invention;

FIG. 6 is a front view of cell culture apparatus including a preferredembodiment of vessel in an associated housing connected to a controlunit;

FIG. 7 is a rear view of the arrangement shown in FIG. 6;

FIG. 8 is a side sectional view of the preferred embodiment of vessel inan associated housing shown in FIGS. 6 and 7;

FIG. 9 is a graph of optical density (at 600 nm) versus time afterinnoculation (in minutes) of uninduced E. coli culture in a 500 mlbottled shaker flask and a 50 liter vessel according to the invention;and

FIG. 10 is a graph similar to FIG. 9 showing optical density of E. coliculture induced with IPTG at OD 0.65 versus time for a cell culture in a500 ml bottled shaker flask and a 50 liter vessel according to theinvention.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 3 show a preferred embodiment of a cell culture vessel 10according to the invention, containing cell culture medium 34.

The vessel has a back wall 21, a front wall 23, two side walls 25, a topwall 27 and a bottom wall 31 constituting a base of the vessel.

Front wall 23 has a lower portion 46 inclined at about 20° to vertical.The front wall has an upper portion including a lower part 26 inclinedat about 60° to vertical and an upper part 28 which is substantiallyvertical.

The back wall 21 includes a lower part 22 that rises vertically and anupper part 24 that is inwardly inclined at about 32°, constituting anabutment that comprises means for redirecting rising gas.

The vessel contains culture medium 34 which has a top surface 32, thevolume of the vessel filled with medium constituting the working volumeof the vessel. A headspace 29 is provided above the medium surface.

The lower chamber portion is defined as being between the lower portion46 of the front wall and the back wall. The upper chamber portion isdefined as being between the upper portion of the front wall and theback wall, including the inclined upper part of the back wall beneaththe surface of the medium.

The vessel has an overall height of 55 cm and a length at the base of 20cm. The upper chamber portion has a maximum width of about 22 cm. Thevessel has an aspect ratio of about 2 and a G value of about 2,calculated as set out above.

A gas inlet means 14 consists of a porous ceramic tube having an insidediameter of 3 mm and a wall thickness of 2 mm with a pore size range offrom 0.1 to 100 microns. The gas inlet means 14 is provided within thelower chamber portion close to the base of the vessel, extending acrosssubstantially the entire length of the vessel base. An associated inlettube 15 leads to the exterior of the vessel, and is provided with anassociated filter assembly 40.

Additional inlet ports 36, 37 are provided for addition of liquidreagents and/or gases. A pressure regulation valve 38, set to operate at2 psi gauge, is provided on the top of the vessel.

A temperature regulation plate 42 is provided slightly spaced from theback wall lower part 22.

A sensor 44 is provided on the lower part 26 of the upper portion of thefront wall, which can be used to monitor operating parameters.

The walls of the vessel are made from two layers of co-extruded nylonand polyeshylene with the nylon on the outside, and is free frommaterials derived from animal sources. The vessel is supported by anouter frame (not shown) or may be a support made of steel, glass orother appropriate materials. The vessel is intended to be disposed ofafter use and a similar fresh vessel can be used for any subsequent cellculture fermentations or mixing operations. The vessel is initiallysupplied in sterile pre-validated condition, sealed in packaging.

In use, sterile gas 30 is introduced continuously to gas inlet means 14via inlet tube 15 from a supply (not shown) at a rate of e.g. 1 literper minute. Gas bubbles with a size range of from 0.1 to 2 mm areproduced and the liquid medium above the gas inlet means 14 risesvertically generally in the direction of arrows 16 (FIG. 1). The risingliquid medium and gas bubbles eventually impinge on the abutmentprovided by inwardly sloping upper part 24 of the back wall. Liquidmedium and gas bubbles are redirected generally in a substantiallyhorizontal direction as represented by arrows 18.

This flow at the culture surface 32 increases the rate of mixing and gastransport from the headspace 29 into the medium 34. Thereafter some ofthe medium circulates within the upper chamber portion about ahorizontal axis as indicated by arrows 18 and 19. Some of the mediumflows downwards into the lower chamber portion in a downcomer asindicated by arrows 48, eventually rising again generally in thedirection of arrows 16. Thus a tubular circulation pattern isestablished within the upper chamber portion parallel to the back wall21.

This arrangement has improved overall aeration and mixing efficiencysignificantly over other arrangements. It can also be seen fromexperimental observations that there is effective mixing within theupper chamber, tower chamber, between chambers and from left to right,thus eliminating dead spots without the requirement for over gassing,allowing the operator to achieve high gas mass transfer and mixing atlow sparging rates.

The use of water miscible dyes to visualize the currents of solutionindicates that there is a highly efficient mixing process in the upperchamber, lower chamber, between the chambers and even from left toright. In experiments, when dye has been introduced at various pointsthroughout the vessel, mixing of an entire vessel, even as large as 50liters, takes only between 2-5 seconds at relatively low aeration flowrates of 2-3 liters per minute.

Liquid additions may be introduced to the medium via inlet ports 36. Anoxygen-enriched sterile air supply, for example, may be introduced tothe headspace via inlet ports 37. In another illustration, it may beadvantageous to introduce an inert gas such as nitrogen or helium if thevessel is being used to mix liquids, dissolve solids into liquids ormaintain suspensions.

An elevated pressure of about 0-2 psi gauge can maintained in theheadspace 29 by the action of the pressure regulation valve 38 with theassociated filter assembly 40. The medium is maintained at any desiredtemperature of up to 42° C. by the temperature regulation plate 42.

The medium conditions are maintained for an appropriate time for thedesired reactions to take place.

FIG. 4 shows a simplified schematic representation of another embodimentof the vessel 50 according to the invention omitting the parts of thevessel above the surface of contained liquid, thus showing only theworking volume.

The back wall 52 is vertical along its full height. The front wall 54 isa continuously inclined wall from top to base at a constant angle ofabout 28° to vertical, bounding both the upper chamber portion and thelower chamber portion. The vessel has an aspect ratio of about 2, a Gvalue of about 2, calculated as set out above, and is asymmetric,

Gas is introduced through elongate gas inlet means 56, positioned nearthe base with minimal distance to either the front or back walls. Gasrises vertically as indicated by arrows 58 and the overall reduction inliquid density in this region causes the liquid also to rise in arrowdirection 58. Once the liquid reaches the surface its movement becomessubstantially horizontal and moves outwards generally in the directionof arrow 60. The liquid circulates generally in the direction of arrow60 until it rises once more generally in the direction of arrows 58.Thus, liquid circulates within the upper chamber portion.

FIG. 5 shows a simplified schematic representation of a furtherembodiment of the vessel 70 according to the invention, similar to thatshown in FIG. 4.

The back wall 72 is vertical along its full height. The front wall has alower portion 74 which rises vertically, a mid portion 76 extendinghorizontally and an upper portion 78 which also rises vertically. Thevessel has an aspect ratio of 2 and a value of about 2, calculated asset out above, and is asymmetric.

Gas is introduced through elongate gas inlet means 80, positioned nearthe base with minimal distance to the back wall 72. The distance to thefront wall 74 is no more than twice the diameter of the gas inlet means80. Gas rises vertically as indicated by arrows 82 and the overallreduction in liquid density in the region causes the liquid also to risein arrow direction 82. Once the liquid reaches the surface its movementbecomes substantially horizontal and moving outwards generally in thedirection of arrows 84. The liquid circulates generally in the directionof arrows 84 until it rises once more generally in the direction ofarrows 82. This liquid circulates in the upper chamber portion.

FIGS. 6 to 8 show cell culture apparatus including a preferredembodiment of a 50 liter vessel 100 in accordance with the inventioncontained in an associated housing 150 and is connected to a controlunit 200 by a gas tube 102.

The vessel 100 is made of transparent flexible plastics material andsits inside the associated housing 150. When filled with liquid thevessel fits snugly and takes the shape of the interior cavity of thehousing 150. The vessel 100 is slightly oversized so that it pushes intothe corners of the housing 150. The overall geometry of the vessel issimilar to that shown in FIGS. 1 to 3 but without tapering sides asshown in FIG. 2.

The housing 150 comprises side walls 152, a rear panel 154, a front wall156, a top portion 158 and a hinged top lid 160. The housing has a sideaccess door 162 in a side wall 152 for access to entry ports (not shown)in the vessel. The vessel is mounted on wheels 164 for ease of transportand use.

The front wall 156 comprises three panels 156 a-c. Each of the panelshaving a transparent window so that the vessel and its contents can beviewed.

The vessel 100 has a height of 105 cm, a width (distance between frontwall 156 c and rear panel 154) of 45 cm and a depth (distance betweenside walls 152) of 62 cm.

The vessel can be accessed by opening the hinged top lid 160. The hingedtop lid is attached to the housing by hinges 166 and is held tightly tothe top of the housing by clips 168. The vessel comprises a gas outlettube 170 with an associated filter 172 which passes through a hole inthe top of the hinged top lid 160 and into the rear of the housing viathe top portion 158.

The control unit 200 is in the form of a box and has a control panel 202on a forward sloping face. The unit contains a computer, which canregulate the flow rate of gas to the vessel via tube 102, as desired.

The control unit receives data during operation of the vessel 100 viadata lines (not shown). Such data includes headspace pressure, pH ofsolution and oxygen saturation. The computer can govern the flow rate ofgas to reach a desired set point in one of these parameters.

EXAMPLE Growth of E. coli

Experimental conditions for all growth media and culture parameters werekept as similar as possible throughout.

Growth media: LB broth (Sigma L3022) was prepared to a concentration of20 g/liter final concentration. Pre induction media was supplementedwith 0.5% glucose to prevent leaky induction of the Lac promoter. Allmedia contained ampicillin (Sigma A9518) to select for the recominantvector.

Cell Line chosen for protein expression was Merck BiosciencesTUNER(DE3)™. This strain was transformed with the recombinant vectorusing the recommended protocol for this strain.

1 liter baffled flasks were used to grow cultures of 500 ml volume.Flasks were agitated vigorously at 225 rpm in a New Brunswick orbitalshaker maintained at

The 50 liter vessel shown in FIGS. 6 to 8 was part filled with deionisedwater which was fed directly into the bag through a Pall Kleenpak filter(Part Number KA3EKVP6G). A concentrate of LB media was added, followedby deionised water to make up the media to the final concentration. A 2liter innoculum was added to the vessel, which had been grown-upovernight in four shaker flasks (500 ml in each).

The vessel according to the invention was used to culture E. coli cellsto high cell densities and gave very comparable growth profiles to thosegenerated in shaker flasks with vigorous agitation (FIGS. 9 and 10).Typically, disposable bioreactors are not used to grow E. coli due tothe difficulties in achieving sufficient oxygen mass transfer, and assuch, this was an excellent experimental model with which to demonstratethe system's efficient aeration process. The results indicate thatgrowth curves and final densities that can be achieved in a 50 litervessel are the same as a 500 ml culture in a flask. In the latter, thechallenges of oxygen mass transfer are minimal.

FIGS. 9 and 10 also demonstrate that a 2 liter flask culture (4×500 ml)can be directly scaled-up to a 50 liter volume with no interim steps—aone-step scale-up was achieved.

The invention claimed is:
 1. A cell culture and mixing vessel forcontaining a liquid, comprising a chamber having a lower chamber portionand an upper chamber portion wider than the lower chamber portion, a gasinlet for supplying gas to the lower chamber portion, the height ofliquid being selectable such that when in use, liquid in the upperchamber portion circulates within the upper chamber portion as a resultof the introduced gas and G =(H ×A)/V is greater than 1.5, wherein A isthe surface area of liquid in the vessel, H is the rise height of thegas and V is the volume of liquid, wherein the chamber has a front walland a back wall, wherein when the back wall is vertically orientated,the front wall is inclined at an angle of incline, and wherein the angleof incline has an initial value which with increasing height isimmediately followed by a reduction in the value of the angle ofincline.
 2. A vessel according to claim 1, wherein the reduction in thevalue of the angle of incline is in turn followed by an increase in thevalue of the angle of incline of the front wall.
 3. A vessel accordingto claim 1, which further comprises an abutment for redirecting risinggas inclined with respect to the direction of rising gas, such that whenin use, rising gas in the form of bubbles, initially rises substantiallyvertically in the liquid and is redirected in a substantially horizontaldirection within the chamber by the abutment.
 4. A vessel according toclaim 3, wherein the chamber comprises a back wall, at least a part ofwhich is vertical or substantially vertical, and a front wall.
 5. Avessel according to claim 4, wherein the abutment is inclined relativeto the vertical or substantially vertical part of the back wall.
 6. Avessel according to claim 5, wherein the abutment is a non-vertical partof the back wall.
 7. A vessel according to claim 1, wherein there is nophysical barrier between any rising regions of liquid and any descendingregions of liquid.
 8. A vessel according to claim 4, wherein the gasinlet extends across substantially the whole length of the back wall. 9.A vessel according to claim 4, wherein at least part of the back wall isplanar, and wherein the at least part of the back wall extending upwardsfrom a level of the front wall at which the front wall has the initialvalue of angle of incline to a level at which the reduction in the angleof incline has occurred.
 10. A vessel according to claim 1, wherein thewidening of the vessel with respect to height occurs asymmetrically. 11.A vessel according to claim 1, wherein the lower chamber portion istapered, becoming wider with increasing height.
 12. A vessel accordingto claim 1, wherein at least a lower part of the upper chamber portionis tapered becoming wider with increasing height.
 13. A vessel accordingto claim 1, which is made out of flexible plastics material.
 14. Avessel according to claim 1, wherein the gas inlet comprises one or moretubes with a gas permeable wall.
 15. A vessel according to claim 1,wherein the gas inlet has a plurality of exit holes, wherein each exithole is of a size in a range of from 0.1 microns to 1 mm.
 16. A vesselaccording to claim 1, further comprising a means for regulating thetemperature of the liquid in the vessel.
 17. A vessel according to claim1, further comprising a means for providing a controllable pressure ingas above the liquid.
 18. A vessel according to claim 1, arranged suchthat when in use, liquid in the upper chamber portion circulates withinthe upper chamber portion as a result of the introduced gas.
 19. Amixing apparatus comprising a vessel according to claim
 1. 20. A mixingapparatus according to claim 19, further comprising a separatecontroller device.
 21. A vessel according to claim 1, wherein thereduction of the angle of incline is by at least 20° .
 22. A vesselaccording to claim 15, in which each exit hole is of a size in a rangeof from 1 to 100 microns.
 23. A method of performing a cell culturefermentation in a vessel, the vessel containing a liquid medium andcells to be cultured, and having a tapered lower chamber portion and atapered upper chamber portion wider than the lower chamber portion, themethod comprising the steps of filling the vessel with a volume V ofliquid medium, thereby to obtain a surface area A of liquid in thevessel, and supplying a gas to the gas inlet of the vessel thereby tocause the gas to be admitted to the lower chamber portion to risethrough a height H to the surface of the liquid and to cause liquid inthe upper chamber portion to circulate within the upper chamber portionas a result of the introduced gas, wherein G =(H ×A)/V is greater than1.5, A being the surface area of liquid in the vessel, H the rise heightof the gas in the liquid and V the volume of the liquid in the vessel.24. A cell culture and mixing vessel, comprising a chamber having atapered lower chamber portion and a tapered upper chamber portion widerthan the lower chamber portion; a gas inlet for supplying gas to liquidin the lower chamber portion; and an abutment with a structuresufficient to redirect rising gas in the form of bubbles, initiallytravelling substantially vertically, substantially horizontally withinthe chamber, wherein G =(H ×A)/V is greater than 1.5, where A is thesurface area of liquid in the vessel, H is the rise height of the gasand V is the volume of liquid, wherein the angle of the taper of thelower chamber portion is in the range of 5° to 40° , the angle of thetaper of at least a lower part of the upper chamber portion is in therange of 25° to 80° , and wherein the lower part of the upper chamberportion has a smaller angle of inclination relative to the horizontalthan the lower chamber portion.